Morphological and molecular characterization of two Trichodina (Ciliophora, Peritrichia) species from freshwater fishes in China

Journal Pre-proof Morphological and molecular characterization of two Trichodina species from freshwater fish in China Zhe Wang, William A. Bourland, Tong Zhou, Hao Yang, Chenxin Zhang, Zemao Gu

PII:

S0932-4739(19)30084-7

DOI:

https://doi.org/10.1016/j.ejop.2019.125647

Reference:

EJOP 125647

To appear in:

European Journal of Protistology

Received Date:

2 February 2019

Revised Date:

17 October 2019

Accepted Date:

23 October 2019

Please cite this article as: Wang Z, Bourland WA, Zhou T, Yang H, Zhang C, Gu Z, Morphological and molecular characterization of two Trichodina species from freshwater fish in China, European Journal of Protistology (2019), doi: https://doi.org/10.1016/j.ejop.2019.125647

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Morphological and molecular characterization of two Trichodina species from freshwater fish in China

Zhe Wanga,b, William A. Bourlandc, Tong Zhoua,b, Hao Yanga,b, Chenxin Zhanga,b, Zemao Gua,b*

a

Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural

University, Wuhan, Hubei, P.R. China, 430070 Hubei Engineering Technology Research Center for Aquatic Animal Diseases Control and

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b

Prevention, Wuhan, Hubei, P.R. China, 430070 c

Department of Biological Sciences, Boise State University MS-1515, 1910 University Avenue,

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*

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Boise, Idaho, USA, 83725-1515

Corresponding author: *Zemao Gu, E-mail: [email protected], Telephone: +86-27-

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87282114, Fax: +86-27-87282114

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Abstract

In the present study, we provide morphological and molecular characterization of two

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Trichodina species, T. acuta Lom, 1970 and T. funduli Wellborn, 1967, isolated from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. Morphological

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characters of the two Trichodina species were mainly investigated on the basis of dry silver nitrate-impregnated specimens. Both species are medium-sized and possess well-developed denticles comprising strongly sickle-shaped blades, well-developed central parts, and straight rays. Trichodina acuta can be easily distinguished from the other Trichodina species that possess a clear central circle by the well-developed sharp blade apophysis, and the gap between ray tip and central circle. Trichodina funduli is a poorly known species that is easily confused with T. heterodentata Duncan, 1977, however the latter species has thinner denticles. The small

subunit ribosomal RNA gene sequences of Trichodina acuta and T. funduli were incorporated into phylogenetic analyses. Our findings suggest that the phylogenetic lineage of trichodinids might not correspond with their living environments, host species or even some morphological characteristics.

Keywords: Fish parasite; Molecular phylogeny; Small subunit rRNA gene; Trichodina acuta; Trichodina funduli; Trichodina hypsilepis.

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Introduction

Trichodina Ehrenberg, 1830 is an economically and ecologically important genus of ectoparasitic or symbiotic protozoan ciliates. To date, about 400 Trichodina species have been

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found on various aquatic animals worldwide in a range of habitats including freshwater,

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brackish water, and marine environments (e.g., Basson and Van As 1992, 2006; Lom 1958; Özer 2007; Tang et al. 2017; Van As and Basson 1989; Xu et al. 2000). Despite universally

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accepted taxonomic criteria, proposed as an effective way to identify and distinguish trichodinids, it is still difficult to discriminate between morphologically similar species partly

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due to the high variation in host species, morphometric data, and some morphological characters (Lom 1958; Tang et al. 2017; Van As and Basson 1989; Wang et al. 2018). In

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addition, most reported Trichodina species are described and identified solely on the basis of morphological characters, without morphometry or molecular data, and many species lack a

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description based on the universally accepted taxonomic criteria. To avoid this conundrum, an integrated approach including detailed morphological characters, host specificity, habitat and molecular data, is increasingly used for identification and phylogenetic exploration of Trichodina species (Gong et al. 2006, 2010; Irwin et al. 2017; Marcotegui et al. 2018; Wang et al. 2017a). In recent years, many studies focusing on Trichodina species have been carried out worldwide (Abdelkhalek et al. 2018; Hu 2012; Mitra et al. 2013; Tang et al. 2017; Wang et al.

2017a, 2018; Zhou et al. 2008). Nevertheless, only a few studies provided both morphological and molecular characterization of Trichodina species. This is partly due to difficulties in the separation and purification of sufficient numbers of confidently identified individuals of a single Trichodina species when mixed infestations may be present (Wang et al. 2017a, 2019). The evolutionary relationships among Trichodina species remain unclear due to the relatively limited number of sequences available in molecular databases (Zhang et al. 2015). It is also worth noting that the sequences of Trichodina species submitted to GenBank need to be verified for taxonomic accuracy since infestations involving multiple Trichodina species are

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common and samples are also easily contaminated by other eukaryotic organisms (Wang et al. 2018).

A project was initiated in 2013 to make a survey of common Trichodina species in central

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China and to build a rapid detection method for Trichodina species (Wang et al. 2017a, 2018,

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2019). In the present study, we provide morphological and molecular characterization of two Trichodina species from China, T. acuta Lom, 1970 and T. funduli Welolborn, 1976, isolated

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from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. The morphological characters and morphometric data of the two species were investigated on the

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basis of traditional dry silver nitrate-impregnated specimens, and sequences of the small subunit ribosomal RNA (SSU rRNA) genes. In addition, the phylogenetic positions of the two

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species and the evolutionary relationships among Trichodina species were analyzed.

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Material and Methods

Sample collection and morphological methods The host fish, koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), were

collected from a koi farm in Xinyang, Henan Province (32°09′17″N, 114°05′52″E) in June 2017, and Xiantao, Hubei Province (30°22′53″N, 113°21′17″E) in May 2018, respectively. Live host fish samples were transported to the laboratory and kept in a tank containing freshwater prior to being anesthetized with MS-222 (tricaine methane sulfonate). Skin

scrapings were placed in Petri dishes containing distilled water. The pure cells were isolated by glass micropipettes under a dissecting microscope. Specimens of Trichodina were taken from each fish specimen and air-dried smears were impregnated with the dry silver nitrate method to reveal structural details of the adhesive disc (Foissner 2014; Klein 1958). The impregnated Trichodina species were observed under a compound microscope at 1000× to check whether the host was infested only by a single species. Measurements and counts of morphological characteristics were done at magnifications of

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600× or 1000×. Drawings were performed with Photoshop CS6 Extended (Adobe Systems Inc) using microphotographs as a template. All measurements of morphological characteristics are expressed in micrometers with the range (mean ± S.D.) following the uniform specific characteristic system proposed by Lom (1958) and Van As and Basson (1989). Detailed

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descriptions of the denticles are presented according to Van As and Basson (1989).

ZooBank registration

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ZooBank registration number of the present work (ICZN 2012, recommendation 8A) is urn:lsid:zoobank.org:pub:2D6BBBD9-0117-4DF9-ACDE-C58EE775293F.

ZooBank

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registration numbers of T. acuta and T. funduli are urn:lsid:zoobank.org:act:A5C65092-B1C64F1D-93D8-5AB4D445EBCC and urn:lsid:zoobank.org:act:3060D7EF-7498-47F0-B84A-

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75D5038E392E, respectively.

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Deposition of voucher material The silver nitrate preparations have been deposited at the Laboratory of Fish Diseases,

College of Fisheries, Huazhong Agricultural University with registration numbers as follows: T. acuta (WZ201707001–WZ201707003) and T. funduli (WZ201805001–WZ201805005).

DNA extraction, polymerase chain reaction (PCR), and sequencing

Genomic DNA was extracted with Trans Direct Animal Tissue PCR Kit according to the manufacturer's instructions (Trans, Bei Jing, China). Reliability of sequences was ensured as follows: the genomic DNA was extracted after confirming the host was infested by a single Trichodina species; the kit used in the present study makes it possible to perform DNA extraction by using 10–20 cells; at least five parallel groups were set up for extracting genomic DNA. The primers Euk A and Euk B (Medlin et al. 1988) were used for amplifying small subunit (SSU) rRNA gene using PCR under cycling parameters as follows: initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing

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at 56 °C for 30 s, and extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min. The PCR products were separated, purified and sequenced by previously reported

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methods (Deng et al. 2015; Guo et al. 2018; Wang et al. 2017b).

Phylogenetic analyses

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In addition to the new sequences, the SSU rRNA gene sequences used for phylogenetic

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analyses were chosen according to Wang et al. (2017a, 2018) and downloaded from the NCBI GenBank database (accession numbers are given in Fig. 5). The chosen sequences were aligned

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by MAFFT v7 and further edited by GBLOCKS (minimum conserved position = 22, minimum flank position = 22, maximum nonconserved positions =8, minimum block = 5, allowed gap

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positions = with half) (Katoh and Standley 2013; Talavera and Castresana 2007). The final alignment including 1636 sites was used for constructing phylogenetic trees using Bayesian

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Inference (BI) and Maximum Likelihood (ML) methods. The GTR+I+G model was the optimal evolutionary model for Bayesian Inference and Maximum Likelihood analyses selected by jModelTest 3.7 under the Akaike information criterion (Darriba et al. 2012). Maximum Likelihood (ML) analysis was performed with RAxML v8.2.10 (Stamatakis 2014) with 1000 replicates. Bayesian inference was carried out using MrBayes 3.2.5 (Ronquist and Huelsenbeck 2003) with 1,000,000 generations sampling every 100 generations. The first 25% of trees were discarded as burn-in. Trees were edited in MEGA 7 (Kumar et al. 2016) or FigTree

(http://tree.bio.ed.ac.uk/software/Figuretree/).

Constrained analyses Four hypothetical topologies including monophyly of: (1) Trichodina species, (2) marine Trichodina species, (3) Trichodina species and Trichodinella species, and (4) Trichodina species with the central circles, ridges or granules were constructed in IQ-TREE v1.6 (Nguyen et al. 2014) with 10000 replicates. The topologies were compared to the non-constrained topology using Approximately-unbiased (AU) test (Shimodaira 2002), Shimodaira-Hasigawa

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(SH) test (Shimodaira and Hasegawa 1999), and Kishino-Hasegawa test (KH) (Kishino and Hasegawa 1989) conducted in IQ-TREE v1.6 (Nguyen et al. 2014) with 10000 replicates.

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Results

Trichodina acuta Lom, 1970 (Figs. 1 a–d, 2a, b; Table 1)

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1960 Trichodina domerguei f. latispina – Lom, Acta Soc. Zool. Bohemoslov. 24, 246, Figs. 1,

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2, 3, 4b, Table 1 (misidentification; desceription of populations from Diaptomus vulgaris, Eudiaptomus gracilis, different species of fish, and tadpoles found in Czech Republic)

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1961 Trichodina domerguei f. acuta f. n. – Lom, Acta Soc. Zool. Bohemoslov., 25: 216, Fig. 1–2 A, D–F (although Lom designated Figs. 1, 2, 4, 5, 8, 9 as figures of this species on

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page 216, these figures are not availuable in this paper), Tables I–II (name not available, for details, see discussion; no type material available).

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1963 Trichodina domerguei f. latispina Dogiel, 1940 – Chen, Acta Hydrobiol. Sin., 3: 99, Fig. 1, Table 1 (misidentification; record of populations from Cyprinus carpio, Hypophthalmichthys molitrix, tadpoles, Sinodiaptomus sasii, abd Neodiaptomus handeli found in Wuhan, China). 1968 Trichodina domerguei f. acuta Lom, 1961 – Kazubski and Migala, Acta Protozool., 6: 142, Plates IV–V (Figs 15–26), Table 3 (remarks on the seasonal variability of T. acuta). 1968 Trichodina domerguei f. acuta Lom, 1961 – Stein, Acta Protozool., 5: 237, Plate II7

(description of a population found in European part of USSR). 1970 Trichodina acuta Lom, 1961– Lom, Arch Protistenkd., 112: 154 (original description of Trichodina acuta based on the population designated as "T. domergui f. acuta" by Lom 1961). 1977 Trichodina acuta Lom, 1961 – Duncan, Trans. Am. Microsc. Soc., 96: 76, Fig. 1 (incorrectly dated; description of a population from Nueva Eciia, Philippines). 1980 Trichodina acuta Lom, 1970 – Jusupov and Urasbaev, Parazitologiya, 14: 505, Fig. 2, Table 2 (incorrectly dated; description of a population from Eastern European

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population).

1983 Trichodina acuta Lom, 1961 – Basson, Van As and Paperna, Syst. Parasitol., 5: 246, Fig. 2 (incorrectly dated; description of a population from Israel).

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1989 Trichodina acuta Lom, 1961 – Albaladejo and Arthuer, Asian Fish Sci., 3: 3, Fig. 1, Table 1 (incorrectly dated; description of a population from Indonesia).

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1989 Trichodina acuta Lom, 1961 – Van As and Basson, Syst. Parasitol., 14: 163, Fig. 3D,

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Table 1 (incorrectly dated; detail description of denticle morphology based on the method proposed by Van As and Basson (1989); description of constant characteristics of

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different populations).

1993 Trichodina acuta Lom, 1961 – Basson and Van As, Acta Protozool., 32: 102, Figs. 1–2,

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Table 1 (incorrectly dated; description of a population from South Africa; reference material: slide 87/07/22-03 in the collection of authors).

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1993 Trichodina acuta Lom, 1961 – Grupcheva and Sedlaczek, J. Appl. Ichthyol., 9: 123, Fig. 2 (incorrectly dated; description of a population from Sachsen, Germany).

1994 Trichodina acuta Lom, 1961 – Basson and Van As, Syst. Parasitol., 3: 200, 217, Figs. 9C–D, 10B, Table 1 (incorrectly dated; detail description of denticle morphology of a population from Taiwan, China; reference material: slide 88/10/11-76 in the collection of authors). 1998 Trichodina acuta Lom, 1961 – Gaze and Wootten, Folia Parasitol., 45: 178, Fig. 2A–G,

Table 1 (incorrectly dated; description of seven populations from Israel; record of the smallest specimen size of T. acuta). 2003 Trichodina acuta Lom, 1961 – Vera, Simonović, and Vesna, Acta Vet. (Beograd), 53: 42, Fig. 1b (incorrectly dated; description of the preference of T. acuta infesting carp; description of species is lacking). 2004 Trichodina diaptomi Šrámek-Hušek, 1953 – Asmat, Pak. J. Biol. Sci., 7: 2066, Fig. 1A (misidentification; desceription of a population from Kalyani of Nadia District, West Bengal, India).

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2005 Trichodina cf. acuta Lom, 1961 – Dove and O’Donoghue, Acta Protozool., 44: 54, Fig. 4 (incorrectly dated; description of a population from Walkamin Research Station, North Qld., Australia).

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2006 Trichodina acuta Lom, 1961 – Tao and Zhao, Acta Zootaxon. Sin., 31: 786–787, Figs.

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4, 11 (incorrectly dated; description of denticle morphology of a population from Chongqing, China).

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2010 Trichodina acuta Lom, 1961 – Basson, Acta Protozool., 49: 259, Figs. 8, 9, 15, Table 5 (incorrectly dated; detail description of denticle morphology of a population from

authors).

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Tasmania, Australia; reference material: slide 98/02/23-01 in the collection of the

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2010 Trichodina acuta Lom, 1961 – Kibria, Islam, Habib, and Asmat, Wiad. Parazytol., 56: 155–157, Figs 3, 7–9, Table 1 (incorrectly dated; description of a population from

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Gazipur, Bangladesh; reference materials: slide MB 1 and MB 2, deposited in Museum of Department of Zoology, University of Chittagong, Chittagong 4331, Bangladesh).

2011 Trichodina acuta Lom, 1961 – Qi, Zhao, and Tang, J. Chongqing Uni., 28: 19, Figs. 3C, 4D (incorrectly dated; description of a population from Sichuan, China). 2011 Trichodina acuta Lom, 1961 – Han and Zhao, J. Neijiang Uni., 26: 23, Figs. 1B, 2B (incorrectly dated; description of a population from Sichuan, China; new host record). 2013 Trichodina acuta Lom, 1961 – Mitra, Bandyopadhyay, and Gong, Parasitol. Res., 112:

1080, Figs. 13, 25 (incorrectly dated; description of a population from India). 2017 Trichodina acuta Lom, 1961 – Wang, Zhou, Yang, and Gu, Eur. J. Protistol., 60: 52, Fig. 1, Table 1 (incorrectly dated; description of a population from Hubei, China; analysis of SSU rRNA sequence KX904932; one silver nitrate impregnated slide deposited at the Laboratory of Fish Diseases, College of Fisheries, Huazhong Agricultural University with registration number DQ2013051901). 2019 Trichodina acuta Lom, 1961 – Wang, Zhou, Yang, and Gu, Aquac. Res. 50: 3276, Figs.

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1a–c, 2a–c, Table 1 (incorrectly dated; description of a population from Hubei, China).

Description of Henan population (n = 20): The morphometric data is shown in Table 1. Denticles fitting tightly to each other. Blade well developed, broad, strongly sickle-shaped with

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a sharp tangent point, filling space between y-axes. Distal blade surface smooth, at same level

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as, or slightly above tangent point. Blade apophysis well developed, angular. Posterior projection inconspicuous or well developed and angular, fitting tightly into blade apophysis of

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preceding denticle. Anterior margin of blade smooth, slightly sloping towards prominent blade apex that touching or slightly extending beyond y-axes. Posterior margin of blade forming deep

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curve, deepest point lower than blade apex. Section connecting blade and central part well developed, sturdy. Blade connection well developed and strong. Central part well developed

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and squat, extending slightly more than half way towards y–1 axis. Central part above and below x axis with slanting distal edge, almost similar in shape. Ray connection strongly

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developed, thick. Ray robust, becoming thicker after ray connection, keeping equal thickness for most of length, becoming narrower towards sharp tip. Ray straight, nearly parallel or forwards to y-axis. Ray apophysis inconspicuous in some specimens (Fig. 2a), in most specimens well developed and round, fitting closely into lower central part indentation of follwoing denticle (Fig. 2b). Central zone of disk with an irregular circle impregnated more lightly than rest. Adoral ciliary spiral turns about 390° around peristomial disc. Nuclear apparatus insufficiently impregnated.

Host and site: Cyprinus carpio, on the skin of tail fin. Locality: Xinyang, Henan, Hubei province, China (32°09′17″N, 114°05′52″E). Prevalence: 4/4 (100%). GenBank number: MK757999.

Trichodina funduli Wellborn, 1967 (Figs. 3a–f, 4a, b; Table 2) 1967 Trichodina funduli n. sp. – Wellborn, J. Protozool., 14: 401, Figs. 5, 22 (original description; type materials: USNM Helm. Coll. No. 61645 and 61646.).

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1967 Trichodina hypsilepis n. sp. – Wellborn, J. Protozool., 14: 401, Figs. 8, 27 (original description of synonym; type materials: USNM Helm. Coll. No. 61651 and 61652).

1967 Trichodina salmincola n. sp. – Wellborn, J. Protozool., 14: 405, Figs. 14, 26 (original

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description of synonym; type materials: USNM Helm. Coll. No. 61657 and 61658).

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1970 Trichodina funduli Wellborn, 1967 – Lom, Arch. Protistenkd., 112: 168 (first revison of T. funduli; tentative proposal of T. hypsilepsis and T. salmincola as subspecies of T.

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funduli).

1984 Trichodina hypsilepis Wellborn, 1967 – Arthur and Lom, Trans. Am. Microsc. Soc., 103:

available).

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175, Figs. 7–8, Table 1 (description of a population from Cuba; no reference material

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2007 Trichodina hypsilepis Wellborn, 1967 – Gong, Dissertation, p. 48, Fig. 3.2B (description of a population isolated from tadpole of Rana sp. from Hubei, China; analysis of SSU

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rRNA gene sequence EF524274). 2019 Trichodina hypsilepis – Jiang, Wang, Xiong, Yang, Sun, Feng, Warren, and Miao, Mol. Phylogenet. Evol., 132: 26 (genomic-scale data of T. hypsilepis).

Description of Hubei population (n = 40): The morphometric data is shown in Table 2. Medium-sized trichodinid. Denticles fitting tightly to each other. Blade well developed, broad, strongly sickle-shaped with a sharp tangent point, filling space between y-axes. Distal blade

surface smooth, at same level as, or above tangent point. Blade apophysis inconspicuous or well developed, angular. Anterior margin of blade smooth, slightly curving towards to prominent blade apex. Blade apex always extending beyond y-axes. Posterior margin of blade forming deep curve, deepest point lower than blade apex. Section connecting blade and central part well developed, sturdy. Central part well developed and conical, extending slightly more than half way towards y–1 axis. Posterior projection absent. Blade connection developed. Central part above x axis with slanting distal edge, while central part below x-axis with straight edge that almost parallel to x-axis. Ray connection strongly developed, thick. Ray robust and

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long, becoming narrower towards round tip. Ray straight, nearly parallel or forwards to y-axis. Ray apophysis absent or inconspicuous in some cases (Fig. 4a), in most specimens round and fitting slightly into lower central part indentation of following denticle (Fig. 4b). Macronucleus

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horseshoe-shaped, situated centerally. Micronucleus spherical, situated in -y1 position. Adoral

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ciliary spiral turns about 390°–410° around peristomial disc.

Host and site: Paramisgurnus dabryanus, on gills in particular, and on skin of body

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surface and fins in general.

Locality: Renrui farm, Xiantao, Hubei province, China (30°22′53″N, 113°21′17″E).

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Prevalence: 29/29 (100%).

Mean intensity and range: 19 ± 20; 3–78.

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GenBank number: MK757998.

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Phylogenetic analyses of the SSU rRNA gene sequences The length and GC content of the SSU rRNA gene sequence of T. acuta are 1624 bp and

51.05% respectively. The most similar species by BLAST in GenBank were: a population of T. acuta (KX904932, 99.9%, 1620/1623bp), T. heterodentata (AY788099, 96.9%, 1576/1626 bp), and T. hyperparasitis (KX904933, 96.9%, 1574/1625 bp). The length and GC content of the SSU rRNA gene sequence of T. funduli are 1605bp and 48.35%, respectively. The most similar species by BLAST in GenBank were: T. hypsilepis (EF524274, 99.9%, 1603/1606 bp), T.

bellottii (MH730162, 94.9%, 1508/1589 bp), and T. heterodentata (AY788099, 91.2%, 1479/1622 bp). The phylogenetic tree (Fig. 5) was constructed based on the topology of Maximum Likelihood (ML) tree because the Maximum Likelihood (ML) tree and Bayesian Inference (BI) tree showed almost congruent topology. The tree showed that the Urceolariidae, comprising the genera Urceolaria and Leiotrocha, grouped in a clade (84/0.91) as sister to the Trichodinidae. Trichodinidae comprising Trichodina species and Trichodinella species formed a fully supported (100/1.0) monophyletic clade. A Trichodinella species was nested within

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Trichodina species. Our population of T. acuta was nested within a clade comprising freshwater Trichodina species. Our population of T. funduli, a previously described population of T. hypsilepis (EF524274), and T. bellottii formed a clade which was sister to all other species of

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Trichodinidae.

Constrained analyses

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Log-likelihood and p-values for constrained analyses are shown in Table 3. The SH test did not reject the monophyly of freshwater Trichodina species and the monophyly of marine

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Trichodina species (p > 0.05), whereas monophyly of both was rejected by the AU and KH tests (p < 0.05). Based on the result of the AU test, the four hypothesized monophylies can be

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strongly rejected.

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Discussion

Nomenclature and taxonomy of Trichodina acuta Based on the morphological structure of the adhesive disc, we consider the Trichodina

isolated from the skin of Cyprinus carpio in the present study to be conspecific with T. acuta. Since it was first mentioned by Lom in 1961 as a new form of T. domergueii Wallengren, 1897, T. acuta has been found worldwide, mainly on the skin of freshwater fish (Basson 2010; Lom 1961; Tao and Zhao 2006; Wang et al. 2017a). Based on the description of Raabe (1959), all

species with a clear central circle in the adhesive disc were long assumed to belong to an infrasubspecific taxon (form) of T. domergueii (Lom 1961). However, the validity of this form has been questioned since the dry silver nitrate impregnation was introduced for studying trichodinids (Arthur and Lom 1984; Lom 1970). According to Articles 10.2 and 15.2 of the ICZN 1999, a new name published after 1960 as a “variety” or “form” is considered as infrasubspecific and thus is not regulated by the code. The name “Trichodina domerguei f. acuta f. n.” in Lom (1961) and is, thus, unavailable. In addition, “nov. comb.” as applied to Trichodina acuta by Lom (1970) is also incorrect because the generic classification was

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unchanged. Consequently, the species T. acuta was first made available by Lom (1970) and thus the correct name is Trichodina acuta Lom, 1970. Most of the subsequent studies used the incorrect date 1961 (see synonyms list of T. acuta), thus the correct date 1970 should be used

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instead in future studies. Additonally, it was noted that T. domergueii was first decirbed by Wallengren in 1897, however almost all the subsequent studies used the wrong name

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“Trichodina domerguei” (Asmat 2004; Chen 1963; Irwin et al. 2017;Lom 1960; Wallengren

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1897).

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Based on the results of the following studies, T. acuta is larger than T. domergueii in terms of overall body dimensions. Additionally, the ray tip of T. acuta is sharp, whereas that of T.

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domergueii is round (Fig 2c) (Grupcheva and Sedlaczek 1993; Irwin et al. 2017; Özer 2003). Basson et al. (1983) reported two populations of T. acuta: one from South Africa and the other

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one form Israel. Later, Van As and Basson proposed that the population from South Africa, previously described as T. acuta, and some new records from South African should all represent a new species, T. compacta (Van As and Basson 1989). According to Van As and Basson (1989), characteristics of the adhesive disc should be the most important differences between T. acuta and T. compacta. Compared with T. compacta, the central circle of T. acuta occupied a smaller area of the adhesive disk and lacks the well-defined periphery (Fig 2a, b, d). In addition, there is a distance between the central circle and the ray tips of T. acuta, whereas the ray tips of T.

compacta are closely associated with the periphery of central circle (Fig 2d). Thus, the Trichodina species isolated from the skin of C. carpio in the present study was considered to be conspecific with T. acuta. The molecular analyses also support this view. It is worth noting that T. acuta in the present study and the previous population we reported have fewer denticles than the populations found in other places in China but falls within the expected range of morphometry (Table 1) (Basson 2010; Gaze and Wootten 1998; Han and Zhao 2011; Tao and Zhao 2006; Wang et al. 2017). We infer that this discrepancy may be due to different environments, host species, and the relatively high intraspecific variability of T. acuta (Gaze

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and Wootten 1998; Kazubski and Migala 1968).

In earlier studies, some populations of T. acuta isolated from fish were described as T. domerguei f. latispina Dogiel, 1940 and T. diaptomi Šrámek-Hušek, 1953 (Asmat 2004; Chen

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1963). Dogiel (1940) first described a trichodinid, namely T. domerguei f. megamicronucleata, isolated from a Diaptomus species. He described five other trichodinids from fish, including T.

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domerguei f. latispina in the same paper. Later, Šrámek-Hušek (1953) described a trichodinid

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namely Trichodina domerguei var. diaptomi isolated from Diaptomus vulgaris. However, no photomicrographs of silver nitrate-impregnated specimens of above metioned species from

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Diaptomus species are available. Lom (1960) presented the photomicrographs of silver nitrateimpregnated specimens of T. domerguei f. latispina, and he concluded the trichodinids

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described by Dogiel (1940) should be divided into two groups: the trichodinids infesting fish should be synonyms of T. reticulata, and the trichodinids infesting diaptomids (Copepoda)

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should be synonyms of T. domerguei f. latispina. After re-evaluating the host specificity and comparing the denticle morphology, Basson and Van As (1991) concluded that all the trichodinids infesting planktonic copepods should represent the same species, namely T. diaptomi. They also indicated that high host specificity of T. diaptomi precluded frequent infestation of fish. Thus, based on the views raised by Basson and Van As (1991) and the morphological characters, T. domerguei f. latispina described by Chen (1963) and T. diaptomi described by Asmat (2004) are most likely T. acuta. It is noted that Chen (1963) also described

a population of T. domerguei f. latispina from Sinodiaptomus sarsi and Neodiaptomus handeli and he succeeded in transferring this population from calanoids to carps. Basson and Van As (1991) thought this population should also be T. diaptomi. However, Chen’s (1963) establishment of a viable population on fish of trichodinids from copepods is the only such instance, further study, including molecular and morphological methods of this Trichodina species, should be carried out to ascertain whether it should be T.acuta, T. diaptomi, or a new

Nomenclature and taxonomy of Trichodina funduli

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species.

On the basis of morphological characters of the denticle, the Trichodina isolated from the gills and body surface of Paramisgurnus dabryanus closely resembles T. hypsilepis. The

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population in the present study has a smaller cell diameter and shorter rays than those of

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populations described in previous studies, but the other morphological characters are quite similar (Arthur and Lom 1984; Gong 2007; Wellborn 1967). Based on the structure of the

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denticles and the results of molecular analyses, we considered the Trichodina species we collected from the loach to be conspecific with T. hypsilepis. Wellborn (1967) first described

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four similar Trichodina species: T. davisi from Morone saxatilis, T. funduli from Fundulus notti, T. hypsilepis from Notropis hypsilepis, and T. salmincola from Salmo gairdneri and Salvelinus

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fontinalis from the USA. As noted by Lom (1970) and Arthur and Lom (1984), despite the variable position of the micronucleus (+y position for T. hypsilepis and T. davisi and -y1

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position for T. funduli and T. salmincola), the other morphological characters and morphometric data of these four Trichodina species are, in all cases, quite similar and conspecificity cannot be excluded. In the present study, we present the denticle morphological charateristics of these species based on methods proposed by Van As and Basson (1989). The results show that the denticle morphology of T. funduli, T. hypsilepis and T. salmincola is almost the same and thus these trichodinids are likely conspecific. The population of T. hypsilepis described in the present study shows a different position of the micronucleus from the original

description, supporting the opinions that the three Trichodina species described by Wellborn (1967) should be conspecific. According to Article 24 of the ICZN 1999, Lom (1970) should be the first revisor of the three species, thus the name Trichodina funduli should be the valid name. Although T. hypsilepis has been more often used than T. funduli in previous studies, we suggest that T. hypsilepis and T. salmincola be considered synonyms of T. funduli. Although T. davisi was also regarded as conspecific with T. funduli in previous studies (Arthur and Lom 1984; Lom 1970), the denticle characteristics of T. davisi are different from those of T. funduli. The main differences are the characteristics of the blade. In the case of T.

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davisi, the blade is not prominent sickle-shaped and its apex is not prominent (Fig. 4c), whereas the blade of T. funduli is more developed and strongly sickle-shaped. It is not common for a Trichodina species to display such a high variability in blade shape. Therefore, we believe that

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further studies are needed to show whether T. davisi is a synonym of T. funduli.

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Morphologically, T. funduli is similar to T. heterodentata Duncan, 1977. The two species can be distinguished from each other by the following characteristics: T. funduli has more highly

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developed denticles with broader blades and more robust rays, whereas T. heterodentata has narrow denticles (Asmat 2004; Wang et al. 2019). The two species also have divergent SSU

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rRNA gene sequences (Fig. 5).

Molecular data for T. funduli was later provided by Gong et al. (2006). However, the

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morphological data of this population, isolated from unidentified Rana species, was incomplete (Table 2). We measured the single specimen of T. funduli of Gong (2007) and the results are as

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follows: border membrane 6.4 μm in width; denticle 18.5 μm in span, 9.8 μm in length; blade 5.3 μm in length; central part 2.5 μm in width; ray 10.7 μm in length, which are bigger or longer than those of our population. The result suggests that the morphometric variability may exist in different populations of T. funduli.

Phylogeny of T. acuta and T. funduli

Based on the phylogenetic analyses, a clade comprising T. funduli and T. bellottii is sister to the remainder of the Trichodinidae clade, and Gong (2007) inferred that the evolutionary relationships among Trichodina species may be correlated with the type of host. However, our result does not support this opinion, and it is possible that amphibians serve as temporary hosts of T. funduli (Arthur and Lom 1984; Pala et al. 2018). To date, no species of Trichodina exclusively parasitizing amphibian larvae has yet been recorded, indicating the need for a broader investigation of host specificity in this Trichodina group. As noted by Tang et al. (2013), the phylogenetic relationships among Trichodina species can be reflected by the blade

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morphology. The present T. acuta population resides in a clade mostly comprising Trichodina species having sickle/arc-shaped blades, supporting the view metioned above to some extent. However, as more molecular data of Trichodina species have accumulated in GenBank, this

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view has been challenged. Irwin et al. (2017) reported a population of T. domergueii (arc-

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shaped blade) clustered within the clade comprising Trichodina species with quadrangularshaped blades. The phylogenetic positons of T. funduli and T. bellottii also revealed that the

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shape of the denticle blade can’t be regarded as a phylogenetic characteristic. Gong et al. (2006) suggested the granules in the adhesive disk might be a phylogenetic character within

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Trichodinidae. However, constrained analyses in the present study show that the Trichodina species with central circles, ridges and granules do not form a monophyletic group, this

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character may be the result of convergence. The genus Trichodinella can be morphologically differentiated from Trichodina by its

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degenerated rays and 180°–270° turns of adoral ciliary. The present and previous phylogenetic trees all shows that Trichodinella was nested within Trichodina, which revealed that genus Trichodina might be non-monophyletic. Our result of AU test also support this view. However, the placement of genus Trichodinella in the present study may be an artifact and its phylogenetic position should be disscussed based on multigenes. Addtionally, the validity of sequences of Trichodinella species in GenBank should be vertified in future study.

The identification of Trichodina species based solely on morphological and morphometric characters revealed by silver nitrate-impregnation requires considerable expertise and is subject to misinterpretation (Kreier 2013; Tang et al. 2017; Van As and Basson 1989; Wang et al. 2018). Consequently, some persisting taxonomic confusion needs to be resolved. To help address the conundrum mentioned above, molecular sequences, such as small subunit (SSU) ribosomal RNA gene sequence, have been increasingly used, in conjunction with traditional morphologic characterization, as markers to identify Trichodina species (Tang et al. 2017; Wang et al. 2017a, 2018). Our results suggest that phylogenetic relationships among trichodinids might have less

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correlation with habitats, type of hosts or certain morphological characteristics than previously supposed (Irwin et al. 2017; Wang et al. 2017a, 2018). Wider taxon sampling and multigene trees, including transcriptomic and whole genome analyses, will undoubtedly refine our

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understanding of phylogenetic relationships in the Mobilida in general and the Trichodinidae

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in particular.

Authors contributions

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Wang Z. performed the most of the experiments, analyzed the data, arranged the micrographs, and drew the illustrations. Wang Z. and Zhang C. collected the samples. Zhou T.

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and Yang H. helped with most of the experiments. Gu Z. was the manager of the funds. Wang Z. and Bourland W. A. wrote the manuscript and all authors revised the manuscript.

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Acknowledgements

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We are grateful to Associate Editor, Helmut Berger and two anonymous reviewers for their constructive comments that improved the quality of the manuscript. This study was supported by China Agriculture Research System (project No. CARS-46) and Demonstration of Key Techniques for High-Quality Aquatic Products Research (project No. 2016620000001046).

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Figure legends

Fig. 1a–c. Photomicrographs of Trichodina acuta after dry silver nitrate preparations. a–c:

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slightly different adhesive discs of specimens. d: Adoral ciliary spiral turns.

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Fig. 2a–d. Schematic drawings of the denticles of three Trichodina species after dry silver

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nitrate preparations. a, b: Trichodina acuta, Hunan population (originals). c: Trichodina domergueii (after Özer 2003). d: Trichodina compacta (after Van As and Basson 1989). cs, central circle.

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Fig. 3a–f. Photomicrographs of Trichodina funduli after dry silver nitrate preparations (a–d) and from life (e, f). a–c: Silver nitrate-impregnated adhesive disc. d: Adoral ciliary spiral turns. e: Haplokinety (double arrowhead), polykinety (arrowhead), and cytopharynx (arrow). f: Horse-shoe shaped macronucleus (arrow) and spherical micronucleus (arrowhead).

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Fig. 4a–i. Schematic drawings of the denticles of Trichodina species after dry silver nitrate preparations. a, b: Trichodina funduli, Hubei population (originals). (c) Trichodina davisi (after Wellborn 1967). d: Trichodina funduli (after Wellborn 1967). e: Trichodina hypsilepis (after Wellborn 1967). f: Trichodina salmincola (after Wellborn 1967). g, h: Trichodina hypsilepis (after Arthur and Lom 1984). i: Trichodina hypsilepis (after Gong 2007).

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Fig. 5. Phylogenetic tree constructed by SSU rRNA gene sequences. Sequences investigated in the present study are in bold. Numbers given at nodes of branches are the bootstrap percent

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(BS) and posterior probability (PP) values. The scale bar corresponds to 5 substitutions per 100 nucleotide positions.

Table 1 Morphological data of Henan population (present study) of T. acuta and comparison with some other populations Van As Basson Lom

and

Character a

and Van (1961)

Tao and Basson

Wang et

Present

(2010)

al. (2017)

study

58–75

43.6–62.9

48.2–61.3

Zhao,

Basson As (1994)

(2006)

46.3–58.1

50.0–67.0

60.0–70.0

(51.7 ±

(59.0 ±

(64.1 ±

4.0)

4.1)

2.9)

36.5–46.9

39.0–57.0

50.0–60.0

(43.0 ±

(49.4 ±

(56.2 ±

3.8)

4.2)

3.2)

3.6–4.9

4.0–5.0

59–78

Adhesive disc 42–53

membrane

3.5–5

(4.8 ±

0.3)

Denticle ring

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(53.1 ±

(54.6 ±

3.9)

4.5)

3.8)

47–63

35.6–49.6

39.1–50.8

(52.8 ±

(43.2 ±

(45.1 ±

4.2)

3.9)

3.5)

3.9–6.3

3.8–5.5

(4.8 ±

(4.7 ±

0.5)

0.4)

4.0–5.0

5–6 (5.6 ±

(4.4 ± 0.5)

0.3)

0.3)

20.5–30.5

23.0–36.0

28.0–37.0

26–36

23.2－32.8

18.4–28.7

(26.1 ±

(29.7 ±

(33.2 ±

(30.9 ±

(27.0 ±

(23.2 ±

2.8)

3.4)

2.6)

2.4)

2.3)

2.6)

9.3–16.4

8.0-13.0

(13.7 ±

(11.0 ±

1.8)

1.3)

20–23 (21)

18–22 (19)

ur

23–32

diameter

(4.3 ±

na

width

lP

Border

re

diameter

(64.2 ±

-p

Cell diameter

ro of

(1989)

10.5–15

6.3–11.3

Central circle

-

12.0-13.0

(12.7 ±

-

(8.5 ±

diameter 1.2)

1.1)

Denticle 18–21 number

21–25

19–22 (20)

17–20 (18)

17–22 (19)

Radial pins 8

8–9 (9)

7–11 (9)

9–10

9–13 (10)

9–13 (11)

8–11 (9)

13.0–17.0

15.0–18.0

15–19

11.7–16.2

11.8–14.7

(14.3 ±

(16.4 ±

(16.4 ±

(13.6 ±

(13.2 ±

1.1)

0.9)

1.1)

1.2)

0.8)

6.1–7.9

6.0–9.0

8.5–10.0

9.0–11.0

5.7–9.6

6.3–8.8

(7.0 ±

(7.7 ±

(9.4 ±

(9.7 ±

(6.9 ±

(7.7 ±

0.5)

0.7)

0.4)

4.5–6.0

5.0–6.0

per denticle

Denticle span

-

-

Denticle 10–11

2.8–4.4 (3. 4.5–6

(5.4 ± 0.5)

(5.0 ± 0.

(4.5±0.3)

0.4)

0.5)

5) 2.5–4.0

(2.7 ±

(2.8 ±

(3.5 ±

(3.9 ±

(2.8 ±

(3.2 ±

re

2.2–3.6

0.4)

0.4)

0.3)

0.3)

0.4)

3.1–5.6

5.0–8.0

6.0–8.5

6.0–8.5

4.9–7.2

4.5–6.6

(4.2 ±

(6.3 ±

(7.4 ±

(6.9 ±

(6.1 ±

(5.5 ±

0.6)

0.7)

0.6)

0.7)

0.6)

0.5)

na

0.4)

0.8–1.1 0.8–1.0 -

-

-

0.8–0.9

(0.9 ± (0.9± 0. 1) 0.1)

380– 400°

spiral turns

(5.4 ±

3.0–4.5

lP

Adoral ciliary

-

(5.5 ±

3.0–4.0

ur Jo Ratiob

4.0–5.7

2.0–3.5

width

4–7

5.0–6.0

0.6)

1.9–3.2 Central part

Ray length

0.9)

3.7–5.2

5± 0.5)

3–4

0.6)

-p

Blade length

ro of

length

390°

410°

＞360°

390°

390°

Oncorhync Cyprinus Host

Misgurnus Aristichthy

hus

Various

carpio

Cyprinus Tinca tinca

anguillicau

s nobilis

carpio

mykiss

datus

MetsiBohemi Locality

El Rom

matsho

Chongqing

Tasmania

Hubei

Henan

a Reservoir Czech Country

Republi

Israel

China Africa

c

Australia

China

measurements in μm; b Ratio between section of denticle above and below x-axis; “-”, data not

-p

a All

ro of

South

Jo

ur

na

lP

re

available.

China

Table 2 Morphological data of Hubei population (present study) of T. funduli and comparison with some other populations Wellborn

Wellborn (1967)

Wellborn

Arthur and Lom

Character a

Gong (2007) g (1967) c

d

(1967) e

(1984) f 55.1–85.7 (67.4

Cell diameter

70–104 (90)

63–80 (70)

present study

61–85 (74)

49.5–71.4 (59.7 62–77

Adhesive disc

39.8–56.1 (47.6 54–65 (60)

46–57 (57)

40–59 (51)

± 3.9)

± 4.7)

4.1–6.1 (4.9 ±

3.8–5.3 (4.6 ±

4–4.5

width Denticle ring

na

diameter

Denticle number 23–27 (26)

ur

Radial pins per

10

Jo Denticle span

Denticle length

27–35 (32)

21–24 (23)

10

-

0.5)

0.4)

25.5–34.2 (29.4

24.8–36.4 (30.1

29–39 (33)

lP

33–41 (37)

2–5 (3)

re

3–5 (4)

-p

Border membrane

41.6–61.9 (50.4

53–61

diameter

denticle

± 4.7)

ro of

± 6.9)

33–38 ± 2.3)

± 2.7)

20–23 (21.0 ±

21–26 (24)

22–23

22–26

10–12

8–12

0.8)

12

9–12

14.3–19.4 (15.9 -

11–13 (12)

-

-

11–13 (12)

12.3–18.2 (15.0

± 1.0)

± 1.4)

10.2–14.8 (12.4

7.2–10.3 (9.0 ±

10–12 (11)

± 1.1)

Blade length

5–7 (6)

5–6 (5.3)

5–6 (5.8)

4.6–5.6 (5.2 ±

0.8) -

4.1–6.2 (5.2 ±

Central part 2–3 (2.2)

2.0–3.0 (2.6)

0.3)

0.5)

2.0–3.1 (2.5 ±

2.2–4.2 (3.1 ±

2.0

-

width

Ray length

7–10 (9)

7–9 (8)

0.4)

0.4)

6.6–11.2 (8.2 ±

4.9–8.9 (6.7 ±

8–10 (8.5)

0.8)

0.9) 0.7–1.0 (0.8 ±

Ratiob

-

-

-

-

-

ro of

0.1) Micronucleus -y1

+y

-y1

370°

370°–380°

100°

+y

-

-y1

about 390°

380°

390°–410°

position

spiral turns

-p

Adoral ciliary

Host

notti

Notropis

gairdneri,

lP

Fundulus

re

Salmo

hypsilepis

Salvelinus

Unidentified

Paramisgurnus Rana sp.

tadpoles

dabryanus

Country

Alabama

Alabama

North Carolina

The United

The United

The United

ur

Locality

na

fontinalis

Jo

States

a All

States

Havana

Hubei

Hubei

Cuba

China

China

States

measurements in μm; b Ratio betweensection of denticle above and below x-axis; c T. funduli; d, f, g

syn: T. hypsilepis; e syn: T. salmincola; “-”, data not available.

Table 3 Topology tests results based on SSU rRNA gene sequences

Tree

logL

bp-RELL

p-KH

p-SH

p-AU

1 Unconstrained

-11521.57456

0.999

0.998

1

1

2 Trichodina

-11566.64536

0.0003

0.0008

0.152

0.000517

0

0

4.64E-37

Trichodina

with

the

-12116.58436

central circles. ridges or

0

ro of

3

granules 0.0002

5 Trichodina, Trichodinella

-11566.64557

0.0002

0.0016

-p

-11564.76355

0.0008

Jo

ur

na

lP

re

4 Marine Trichodina

0.153

8.54E-05

0.152

0.000556